Topoisomerase II inhibitors, including etoposide and anthracyclines, are among the most effective drugs for the treatment of childhood acute lymphoblastic leukemia (ALL) and are also widely used for breast, lung, ovarian, and testicular cancers. Although ALL is now curable in 70 percent-80 percent of patients, its treatment is complicated by the development of a therapy-related acute myeloid leukemia (t-AML) in up to 12 percent of children who have been cured of their ALL. As this t- AML carries an almost uniformly fatal prognosis, attempts are now being made to limit exposure to topoisomerase II inhibitors (e.g. etoposide and the anthracyclines daunorubicin and doxorubicin), but the success of this empiric strategy and its impact on the efficacy of ALL chemotherapy and the frequency of t-AML remain unknown. T-AML is a distinct clinical and biologic entity, characterized by a unique molecular signature: nonhomologous recombination of the MLL gene with one of a number of partner genes, resulting in leukemogenic genomic fusions. During the last funding period for this grant, we have demonstrated that etoposide can directly induce site-specific nonhomologous recombination in vitro, we have documented recombinogenesis in vivo in children with ALL, and we have developed models for testing recombinogenesis that results as a consequence of topoisomerase II inhibition. We have also identified a clinical genetic host factor, low thiopurine methyltransferase (TPMT) activity, that predisposed patients treated with thiopurines and etoposide to development of topoisomerase II inhibitor-induced t- AML. Subsequently, this genetic polymorphism in TPMT has been linked to t-AML by an independent ALL treatment group, even in children whose only topoisomerase II-inhibitor exposure included the less potent and putatively less leukemogenic anthracyclines. Moreover, we determined that an inherited defect in TPMT significantly predisposes patients to therapy-induced brain tumors. Thus, evidence is mounting that thiopurines contribute to tumorigenesis, particularly in patients with a genetic defect in TPMT. In the continuation of this project, we will use a combination of pre-clinical laboratory and translational clinical studies to (1) determine the contribution of TPMT to etoposide- and anthracycline-induced nonhomologous recombination in isogenic hematopoietic cell lines and in pre-clinical murine models and (2) determine whether the degree of nonhomologous recombination in vivo in children receiving thiopurines and anthracyclines for treatment of ALL is related to their TPMT status. Our hypothesis is that the inopportune concurrence of genetic host factors (such as defective TPMT) and therapy related factors (topoisomerase II inhibitors plus facilitating drugs, such as thiopurines) place a subset of patients at unacceptably high risk of t-AML. Our long- term goal is to identify host- and treatment-related risk factors for t-AML so that we can design less leukemogenic anticancer treatment regimens, with improved efficacy for the primary ALL.